Treatment for dry macular degeneration

ABSTRACT

The present invention relates to altering the physical and/or chemical properties of at least part of at least one tissue in the eye. In a specific embodiment, it relates to the treatment of any eye disorder, although in particular embodiments the individual has a thickened Bruch&#39;s membrane. An activating energy source is utilized to effect a controlled diffusion enhancement and/or degradation of Bruch&#39;s membrane that enables improved diffusional transport between the choroid and retina. The individual is administered an inactivated diffusion-enhancing molecule that becomes associated with the membrane, which is then precisely exposed to an activating energy source, such as light or ultrasound.

The present invention claims priority to U.S. Provisional PatentApplication Ser. No. 60/393,505, filed Jul. 2, 2002, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is generally directed to the fields ofophthalmology and cell biology. Specifically, it relates to altering thephysical and/or chemical properties of an ocular tissue. Morespecifically, it describes a means to detect and/or reduce thethickening and/or change the permeability of Bruch's membrane associatedwith eye disorders, such as macular degeneration. Even morespecifically, it regards administration of an inactivateddiffusion-enhancing molecule to Bruch's membrane followed by activationof the diffusion-enhacing molecule through an energy source.

BACKGROUND OF THE INVENTION

Age related macular degeneration (AMD) is a progressive eye conditionaffecting as many as 10 million Americans. AMD is the number one causeof vision loss and legal blindness in adults over 60 in the U.S. As thepopulation ages, and the “baby boomers” advance into their 60's and70's, a virtual epidemic of AMD will be prevalent. The disease affectsthe macula of the eye, where the sharpest central vision occurs.Although it rarely results in complete blindness, it robs the individualof all but the outermost, peripheral vision, leaving only dim images orblack holes at the center of vision.

Macular degeneration is categorized as either dry (atrophic) or wet(neovascular). The dry form is more common than the wet, with about 90%of AMD patients diagnosed with dry AMD. The wet form of the diseaseusually leads to more serious vision loss.

In the dry form, there is a breakdown or thinning of the retinal pigmentepithelial cells (RPE) in the macula, hence the term “atrophy”. TheseRPE cells are important to the function of the retina, as theymetabolically support the overlying photoreceptors.

The clinical hallmark of atrophic AMD is accumulation of macular drusen,yellowish deposits just deep to the retinal pigment epithelium (“RPE”).Histopathologic examination of eyes with atrophic AMD reveals depositionof lipid and proteinaceous material deep to the RPE in Bruch's membrane.In aged eyes with AMD, Bruch's membrane is often about 3 times thickerthan normal. This thickening is thought to be comprised of lipid as wellas modified and cross-linked protein, which impedes transport ofnutrients across Bruch's membrane from the choriocapillaris to the outerretina. This thickened barrier comprised of lipid and cross-linkedprotein impedes transport of nutrients across Bruch's membrane from thechoriocapillaris to the outer retina. At present, there is no proveneffective treatment for dry AMD other than the use of multivitamins andmicronutrients.

Wet AMD occurs when new vessels form and grow through Bruch's membraneinto the sub-RPE and subretinal space. This neovascular tissue is veryfragile and hyperpermeable. Frequently, it bleeds causing damage to theoverlying retina. As the blood organizes, functional macular tissue isreplaced by scar tissue. To prevent visual loss, it would be desirableto intervene therapeutically prior to the development ofneovascularization.

Although the exact etiology of AMD is not known, several risk factorsseem to be important. For example, ARMD may be caused by chronicexposure of the retina to light. The presence or absence of certainnutrients in the diet, such as the antioxidant vitamins E and C, alsomay affect one's predisposition for ARMD. Other conditions, such ashypertension and smoking, are also considered to be important riskfactors for the development of this disease.

AMD is a challenging disease for both patient and doctor, because thereare very few treatment options and, with the exception of anti-oxidants,no proven preventative therapy. While some individuals experience onlyminor inconvenience from macular degeneration, many others with moresevere forms of macular degeneration are incapacitated. Currenttherapies, including laser photocoagulation, photodynamic therapy, andanti-angiogenic therapeutics have had mixed results, and, in certaininstances, have caused deleterious side effects. A need therefore existsfor a treatment that reduces or limits the effects of maculardegeneration.

Laser photocoagulation is effective in clinical trials, but only aminority of patients with AMD are good candidates for treatment.Furthermore, even after successful ablation of choroidalneovascularization with laser treatment recurrent neovascular tissuegrows frequently. Visudyne® (Novartis Ophthalmics; Duluth, Ga.), aphotodynamic therapy or PDT, uses light-activated drugs to potentiallyhalt or slow abnormal cell growth. The therapy treats late stages ofdisease, characterized by choroidal neovascularization. Briefly, aphotosensitizer is administered intravenously and attaches tolipoprotein receptors, particularly found in cells undergoing rapidproliferation. Shortly after administration, the compound is activatedwith a pre-calculated dose of light at a particular wavelength,resulting in conversion of normal oxygen to free radical singlet oxygen,which in turn causes closure of neovascular tissue. The therapy, inspecific embodiments, treats the blood vessel proliferation. However,because the underlying cause of macular degeneration is not addressed bytreatment of choroidal neovascularization with photodynamic therapy,recurrent neovascularization occurs commonly within several months aftertreatment.

U.S. Pat. No. 5,756,541 is directed to methods to improve visual acuityincluding administering a photoactive compound in an amount sufficientto localize to a target ocular tissue and irradiating the target tissuewith light from a laser, wherein the wavelength of radiation is absorbedby the photoactive compound and the radiation is conducted for a timeand at an intensity sufficient to improve visual acuity. In specificembodiments, the photoactive compound is a green porphyrin. U.S. Pat.No. 5,910,510 is directed to an identical method having a particularirradiation timing.

U.S. Pat. No. 5,798,349 regards methods to treat conditions of the eyecharacterized by unwanted neovasculature, such as AMD, by administeringa liposomal formulation of a green porphyrin in an amount and timesufficient to localize in the neovasculature, followed by irradiation ofthe neovasculature with laser light, wherein the light absorbed by thegreen porphyrin occludes the neovasculature. In the related U.S. Pat.No. 6,225,303, the irradiance is in a range from about 300 mW/cm² toabout 900 mW/cm².

U.S. Pat. No. 6,128,525 is directed to method and apparatus controllingdosimetry of photodynamic therapy.

U.S. Pat. No. 5,935,942 regards methods of occluding vasculature in amammalian eye including co-administering intravenously a fluorescent dyeencapsulated with heat-sensitive liposomes and a tissue-reactive agentactivated by irradiation. The liposomes are heated in the eye to releasetheir contents, wherein the tissue-reactive agent remains inactive,followed by monitoring of fluorescent dye flow within the vasculature.The tissue-reactive agent is activated in the vasculature havingsubnormal blood flow, such that the activated agent chemically occludesthe vasculature. The related U.S. Pat. No. 6,140,314 methods furthercomprise coadministration of a tissue-specific factor effective toimpair growth or regeneration of a blood vessel. The related U.S. Pat.No. 6,248,727 regards related diagnostic reagents and kits.

Thus, although alternative methods for eye disorders exist, the presentinvention addresses a need in the art for therapy for the disorder priorto the point of, for example, neovascularization of the eye tissue,particularly in reversing the pathology of a tissue, such as Bruch'smembrane, associated with the eye disorder.

SUMMARY OF THE INVENTION

The present invention regards methods and compositions for alteringphysical and/or chemical properties of an ocular tissue. In specificembodiments, it refers to enhancement of diffusion through or across atissue, targeted destruction of cells, and/or targeted alteration of atleast part of at least one ocular tissue in an individual. This may beaccomplished, in particular embodiments, using a means to effect acontrolled enhancement of diffusion and/or other controlled alteration,such as with light or ultrasound. Some aspects of the present inventionare directed to treating eye disorders at early stages, and a skilledartisan will recognize the utility of this invention for such a purpose.

In a specific, yet only exemplary, embodiment of the present invention,Bruch's membrane is the targeted tissue. With aging and especially inmacular degeneration, Bruch's membrane develops a lipid and cross-linkedprotein barrier. Impaired diffusion across Bruch's membrane in patientswith macular degeneration, promotes release of angiogenic factors by thenutritionally deprived retina. This, in turn, causes growth ofneovascular tissue through Bruch's membrane with subsequent bleeding,leakage of serous fluid, and severe visual loss. Some aspects of thepresent invention allow treatment/administration before or shortly afterchoroidal neovascularization develops.

To improve diffusion across Bruch's membrane and prevent development ofvisual loss, it is desirable to alter the physicochemical properties ofa tissue associated with visual loss, such as reduce the lipid andcross-linked protein barrier that accumulates in Bruch's membrane withaging and in patients with AMD. This invention is directed to means andcompositions to accomplish this using light or ultrasound to effect acontrolled enhancement of diffusion and/or partial degradation ofBruch's membrane that enables improved diffusional transport between thechoroid and the retina. Energy (exemplary forms being light orultrasound) is utilized to achieve selective activation oftissue-altering substances (which may also be referred to as lipidand/or protein degrading substances), because this use targets thealteration to Bruch's membrane while leaving adjacent tissues minimallyaffected. If active tissue-altering molecules were administeredsystemically, they would not be selective for Bruch's membrane but wouldpotentially damage other tissues. To target the desired tissue (such asBruch's membrane) specifically, the tissue-altering molecule isadministered in inactive form, such as by systemic injection oringestion, or local (intraocular, periocular) injection. In a specificembodiment, the molecule is lipophilic. It binds to multiple tissues inan inactive form before it is gradually eliminated. It is only activatedby an energy source (e.g. light, ultrasound, or both) that is preciselyapplied to the eye to achieve preferential activation of thetissue-altering substance in Bruch's membrane. Once activated, thetissue-altering molecules alter the lipids and/or cross-linked proteinin Bruch's membrane, such as to improve transmembrane diffusion. In aspecific embodiment, to obtain precision in the location of activation,the photochemical activation steps comprise 2-photon photo-chemistry.

In an object of the present invention, there is a method of treating aneye disorder comprising the step of increasing diffusion across Bruch'smembrane in said eye. In a specific embodiment, the increased diffusionis a result of reducing the thickness, altering the composition, orboth, of said membrane.

In an additional object of the present invention, there is a method forincreasing diffusion across Bruch's membrane in at least one eye of anindividual, comprising the steps of administering to the Bruch'smembrane an inactive form of a degradation molecule in an amountsufficient to form a Bruch's membrane/inactive degradation moleculecomplex; and exposing said complex to an activating source, wherein saidactivating source activates said inactive degradation molecule into anactive form of said degradation molecule, said activation resulting inan increase in diffusion across said membrane. In a specific embodiment,the increase in diffusion is a result of reducing the thickness oraltering the composition of said membrane. In another specificembodiment, the increase in diffusion is a result of alteration of alipid, a cross-linked protein, or both in the membrane. In a furtherspecific embodiment, the individual has an eye disorder, such as AMD,juvenile macular degeneration, Sorby's fundus dystrophy, or age-relateddecrease in visual function unrelated to macular degeneration. In aspecific embodiment, the inactive degradation molecule binds directly tothe membrane.

In another specific embodiment of the present invention, the inactivedegradation molecule is a protein, detergent, surfactant (useful forcaged cyclodextrin). In a further specific embodiment, the protein is anenzyme. In an additional specific embodiment, the inactive enzyme isfurther defined as being caged by the incorporation of at least onephoto-removable protecting group on an amino acid sidechain of saidenzyme. In a specific embodiment, the protecting group is o-nitrobenzyl,desyl, phenacyl, trans-o-cinnamoyl, coumarinyl, quinoline-2-onyl,xanthenyl, thioxanthenyl, selenoxanthenyl and anthracenyl, stilbenyl, ora combination thereof. In another specific embodiment, the protectinggroup is o-nitrobenzyl, desyl, phenacyl, trans-o-cinnamoyl, coumarinyl,quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl andanthracenyl, stilbenyl or derivatives thereof. In a specific embodiment,the amino acid is cysteine, aspartate, glutamate, histidine, lysine,asparagine, glutamine, arginine, serine, threonine, tyrosine, or acombination thereof. In a specific embodiment, the detergent is furtherdefined as being caged by a compound comprising at least oneo-nitrobenzyl, desyl, phenacyl, trans-o-cinnamoyl, coumarinyl,quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl andanthracenyl, or stilbenyl group. In another specific embodiment, theprotein is further defined as being caged in an ultrasound contrastagent. In a specific embodiment, the ultrasound contrast agent is amicrobubble or a liposome. In another specific embodiment, the proteinfurther comprises a protein binding domain. In a further specificembodiment, the protein binding domain is a heterodimeric domain. In anadditional specific embodiment, the protein binding domain is a leucinezipper domain, a chitin-binding domain, or a Src homology 2 (SH2)domain.

In another specific embodiment of the present invention, the inactivedegradation molecule is administered to the individual in apharmacologically acceptable composition. In another specificembodiment, the inactive degradation molecule is administered in apharmacologically acceptable composition systemically to the individual.In an additional specific embodiment, the inactive degradation moleculeis administered in a pharmacologically acceptable composition to theindividual orally, by injection (such as periocular or intraocular),rectally, vaginally, or topically. In a specific embodiment, the enzymeis a matrix metalloproteinase, a cholesterol esterase, a lipase, acathepsin, a protease, or a combination thereof. In a specificembodiment, the protease is a serine protease. In a specific embodiment,the activating source is energy. In a further specific embodiment, theenergy is light or ultrasound. In an additional specific embodiment, theexposing step is further defined as exposing said complex to lightenergy from a focused laser source. In another specific embodiment, thedegradation molecule is fluorescently labeled.

In another embodiment of the present invention, there is a method oftreating age-related macular degeneration in at least one eye of anindividual, said macular degeneration characterized by a thickenedBruch's membrane, comprising administering to said individual aninactivated degradation molecule in an amount sufficient for saidmolecule to associate with the membrane to form a Bruch'smembrane/inactive degradation molecule complex; and exposing themembrane to an activating source, wherein following said exposing step,diffusion across the membrane of said eye improves. In a specificembodiment, the method further comprises administering to the individuala fluorescent molecule in an amount sufficient to associate with Bruch'smembrane for visualization of the membrane. In a specific embodiment,the amount of fluorescence emitted is proportional to the amount oflipid or the amount of the altered protein in said Bruch's membrane. Inanother specific embodiment, the wavelength of the light required toexcite the dye, the wavelength of the light emitted by the dye, or thelifetime of the dye is altered in a detectable fashion by the amount oflipid or altered protein in the Bruch's membrane. In an additionalspecific embodiment, the inactivated degradation molecule isadministered in a pharmacologically acceptable composition.

In an additional embodiment of the present invention, there is a kit,housed in a suitable container, comprising an inactivated Bruch'smembrane degradation molecule. In a specific embodiment, the kit furthercomprises an activating source for activation of said inactivatedBruch's membrane degrading molecule. In an additional specificembodiment, the kit further comprises a fluorescent molecule.

In some embodiments of the present invention, a targeted ocular tissueis visualized prior to and/or during activation of the inactivetissue-altering molecule. For example, Bruch's membrane may bevisualized by delivery of a fluorescent molecule that targets Bruch'smembrane; by detecting the autofluorescent signature from characteristiccomponents of Bruch's membrane itself, which differentiates it fromadjacent and surrounding tissues (by well-known means in the art);and/or by using OCT Doppler to identify the membrane's specificmechanical properties (also by well-known means in the art).

In another embodiment of the present invention, there is a method ofdiagnosing an eye disorder in at least one eye of an individual,comprising administering to the individual a fluorescent molecule in anamount sufficient for the fluorescent molecule to associate with Bruch'smembrane in the eye; exposing Bruch's membrane with irradiation to viewthe fluorescence, wherein the quantity of fluorescence is an indicatorof severity of the eye disorder. In a specific embodiment, the eyedisorder is macular degeneration. In another specific embodiment, theirradiation is 2 photon irradiation.

In another embodiment of the present invention, there is a method ofdiagnosing an eye disorder in at least one eye of an individual, basedon the intrinsic light scattering from targeted tissue such as Bruch'smembrane. The technique of optical coherence tomography (OCT) withvisible or infrared light is used to detect alterations in the physicalor chemical nature of Bruch's membrane in the eye. OCT can be used tosee not only the structure in the eye as has been used in some previouswork on the human eye, but can also be used to study the mobility of thestructures by Doppler OCT and the chemical nature by combining the OCTwith an exogenous dye. In this embodiment, the OCT and/or its variantsare used to determine the nature of the Bruch's membrane with alteredproperties to permit guided treatment. Treatment could be thephoto-uncaging or photo-activation or photo-ablation of intrinsic orextrinsic substances in or near Bruch's membrane.

In an additional embodiment of the present invention, there is a methodof treating an eye disorder of an individual, comprising administeringto the individual a visualizing molecule in an amount sufficient topermit visualization of Bruch's membrane in the eye; administering tothe individual an inactive form of a photoactive degradation molecule inan amount sufficient to form a Bruch's membrane/inactive photoactivedegradation molecule complex; and exposing said complex to an activatingsource, wherein said activating source activates said inactivephotoactive degradation molecule into an active form of said photoactivediffusion-enhancing molecule, said activation resulting in an increasein diffusion, alteration of composition, or both, across Bruch'smembrane. In a specific embodiment, the visualization molecule is afluorescent molecule. In another specific embodiment, the fluorescentmolecule is joined to the inactive form of the photoactive molecule. Inalternative embodiments, the membrane is visualized by its signatureautofluorescent properties or by OCT Doppler methods. In an additionalspecific embodiment, the inactive photoactive molecule associates with alipid in Bruch's membrane.

In one embodiment of the present invention, there is a method foraltering an ocular tissue in an individual, comprising the steps ofadministering to the ocular tissue an inactive form of a tissue-alteringmolecule in an amount sufficient to target the molecule to the tissue;and exposing the molecule to an activating source, wherein theactivating source activates the inactive tissue-altering molecule intoan active form of the tissue-altering molecule, the activation resultingin alteration of at least part of the ocular tissue. In a specificembodiment, the method provides therapy for an eye disorder in saidindividual. In another specific embodiment, the eye disorder isage-related macular degeneration, juvenile macular degeneration, Sorby'sfundus dystrophy, age-related decrease in visual function unrelated tomacular degeneration, or glaucoma. In a specific embodiment, theinactive tissue-altering molecule is administered to the individual in apharmacologically acceptable composition. In a further specificembodiment, the inactive tissue-altering molecule is administered in apharmacologically acceptable composition systemically to the individual.In an additional specific embodiment, the inactive degradation moleculeis administered in a pharmacologically acceptable composition to theindividual orally, by injection, rectally, vaginally, or topically. Ifthe administration is by injection, the injection may be intraocular orperiocular, although other routes are acceptable.

In a specific embodiment, optical coherence tomography (OCT) is utilizedfor detection in the targeted tissues or cells, such as detection ofchanges in the composition (such as scattering or labeling with aspecific agent) or the organization of the tissue, an example of whichis Bruch's membrane. Doppler OCT provides informative information, suchas that regarding motility of the scatterers in the targeted tissueand/or regarding targeting of the immobile area (such as the chemicallyaltered area, etc.) for activating them with the uncaging agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates targeted photo-induced tissue alteration of Bruch'smembrane lipids, in an exemplary embodiment of the present invention. Itshows that following systemic administration, caged diffusion-enhancingmolecules diffuse from the choriocapillaris into Bruch's membrane.Two-photon irradiation precisely deprotected the functional groups, andthe diffusion-enhancing molecule is activated specifically withinBruch's membrane.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It will be readily apparent to one skilled in the art that varioussubstitutions and modifications may be made in the invention disclosedherein without departing from the scope and spirit of the invention.

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The term “age-related macular degeneration (AMD)” as used herein isreferred to as macular degeneration in an individual over the age ofabout 50. In one specific embodiment, it is associated with destructionand loss of the photoreceptors in the macula region of the retinaresulting in decreased central vision and, in advanced cases, legalblindness. In specific embodiments, other degenerations are included inthe scope of the term, such as Sorsby's fundus dystrophy.

The term “Bruch's membrane” as used herein refers to a five-layeredstructure separating the choriocapillaris from the RPE.

The term “caged” as used herein refers to the functional groups of atissue-altering molecule being protected by another molecule/moiety. Ina specific embodiment, the term refers to maintaining an inactive formof the tissue-alternating molecule, without an activating source.

The term “drusen” as used herein is defined as yellowish depositslocated deep to the RPE in the inner aspect of Bruch's membrane.

The term “eye disorder” as used herein refers to a condition of havingless than normal health related to at least one eye of an individual. Ina preferred embodiment, the eye disorder comprises a thickened Bruch'smembrane. In a specific embodiment, Bruch's membrane is thickenedapproximately twice its normal thickness. In other specific embodiments,the membrane is thickened about three times to ten times its normalthickness. Specific eye disorders in which Bruch's membrane isabnormally thickened include at least age-related macular degeneration,juvenile macular degeneration, Sorsby's fundus dystrophy, or normalaging with diminished capability to dark adapt. Other eye disorders maynot be characterized by a thickened Bruch's membrane but may benefitthrough alteration of an ocular tissue, such as alteration of trabecularmeshwork to increase outflow facility in glaucoma. Targeted tissuealteration using two photon irradiation can also be used to treatmicrovascular abnormalities in diabetic ocular disease, includingdiabetic macular edema and neovascularization. Selective two photonirradiation and uncaging of inactive drugs following systemic or localadministration can be used to target drug effects to particular tissuesin the eye. This form of selective uncaging can be used in extraoculartissues to achieve selective effects.

The term “macula” as used herein refers to the central area of theretina, including light-sensing cells of the central region of theretina.

The term “macular degeneration” as used herein refers to deteriorationof the central portion of the retina, the macula.

The term “retina” as used herein refers to the neurological tissue atthe posterior of the eye, containing the rods and cones that receivelight and convert it to electrical signals for transmission via theoptic nerve to the brain.

The term “tissue-altering agent” as used herein refers to at least onemolecule that changes the physical, chemical, or both properties of atissue. In a specific embodiment, the term refers to an agent thatalters a tissue such that diffusion through or across is improved, atleast partially. In other specific embodiments, the term refers to anagent that is capable of at least (that at least in part is undesirable)partially degrading components of a tissue. In additional specificembodiments, the term refers to an agent that is capable of reducinglipids and/or cross-linked proteins in a tissue, such as Bruch'smembrane. In specific embodiments, the term regards degrading one ormore of its components. In other specific embodiments, thetissue-altering molecule is a detergent that can extract lipidic andnon-lipidic deposits from within a tissue such as Bruch's membrane.

The term “ultrasound contrast agent” as used herein refers tomicrostructures that can carry exogenous contrast agents. Thesemicrostructures can be disrupted by focused application of ultrasoundirradiation. Examples include microbubbles (tiny gas bubbles, insuspension, that can strongly scatter ultrasound) or liposomes.

II. The Present Invention

The present invention is directed to the treatment of an eye disorder,particularly by effecting alteration of ocular tissue related to thedisorder. This alteration may be of any kind, so long as the tissue isaltered, but in particular embodiments it refers to enhancement ofdiffusion of a tissue using methods and compositions described herein.In a specific embodiment, the methods and compositions affect Bruch'smembrane to improve an ocular disorder.

The eye disorder may be any kind, but in specific embodiments the eyedisorder is AMD or other macular degenerations, such as Sorsby's fundusdystrophy, or any condition that results in thickening of the Bruch'smembrane. In another specific embodiment, age related thickening even inthe absence of macular degeneration is treated. The thickening in theelderly accounts for age related visual changes, such as difficulty indark adaptation. Thus, the methods and compositions of the presentinvention are directed at changing the physical and/or chemicalstructure of an ocular tissue, and in specific embodiments the tissue isBruch's membrane for the improvement of visual function in elderlypatients with or without AMD (and non-elderly patients, such as injuvenile macular degeneration). In specific embodiments, the presentinvention is directed to the treatment of glaucoma with topicaladministration of at least one tissue-altering agent. Such treatment isuseful for glaucoma, such as for the particular embodiment ofalleviating a clogged trabecular meshwork in the pathogenesis ofglaucoma.

In another specific embodiment, the present invention regards treatmentfor macular degeneration, either dry or wet. A skilled artisanrecognizes that wet AMD may be treated with the methods of the presentinvention, given that after treatment of wet macular degeneration bycurrently known methods, the condition commonly recurs (recurrentchoroidal neovascularization). In a further specific embodiment, thetherapy described herein prevents such recurrences and may limit theextent (growth) of existing choroidal neovascularization, thusmaintaining better vision. In specific embodiments of the presentinvention, by increasing nutritional delivery to the retina, thetreatment causes regression of existing choroidal neovascularization.

Thus the present invention aims to treat eye disorders, such as maculardegeneration, by focusing on the thickened Bruch's membrane associatedwith many eye disorders. This thickening is the result of abnormaldeposition of lipid and cross-linked protein and precedesneovascularization through Bruch's membrane, followed by subsequentbleeding, leakage of serous fluid, and severe visual loss. As describedherein, an improvement in diffusion across Bruch's membrane is achieved,thereby reducing the lipid and cross-linked protein barrier thataccumulates in individuals with eye disorders, to prevent development ofvisual loss. In a specific embodiment, the chemical composition of themembrane is altered. For example, a detergent washes away lipids in themembrane. An enzyme degrades proteins within the membrane. In specificembodiments, the methods of the present invention result in an increasein hydraulic conductivity across Bruch's membrane and/or an increase inmacromolecular and/or oxygen permeability of Bruch's membrane.

Generally, an individual with signs or symptoms of an aging Bruch'smembrane is administered, such as systemically or locally, an inactivetissue-altering molecule. In some embodiments, the inactive moleculesare visualizable prior to activation, such as by being fluorescent.Following sufficient time for adequate distribution of the inactivemolecules, the molecules accumulate within multiple tissues, includingBruch's membrane. Once sufficient amounts are reached at Bruch'smembrane, the visualizability of the molecules is used to preciselytarget Bruch's membrane with an energy source, such as light orultrasound, that activates the tissue-altering molecules selectively.

In a specific embodiment, the present invention is useful forvisualization of Bruch's membrane, such as for diagnostic techniques.That is, an inactive fluorescent compound is administered to anindividual and associates with Bruch's membrane, such as by binding alipid in Bruch's membrane. Energy, such as in the form of light, or morespecifically 2 photon irradiation, is focused on the complex of inactivephotoactive compound/Bruch's membrane, and the inactive photoactivecompound is then activated. Energy emitted from the photoactivecompound, such as light, allows visualization of Bruch's membrane. Inspecific embodiments, the amount of light emitted is proportional to theamount of lipid in Bruch's membrane. If the fluorescent molecule isincorporated into the caged tissue-altering agent, visualization ofBruch's membrane can be followed by activation of the degradativesubstance to effect controlled partial degradation of Bruch's membrane.In alternative embodiments, visualization occurs through the naturalautofluorescent activity of constituents of Bruch's membrane and/oroccurs through OCT Doppler methods.

III. Tissue-Altering Molecule

The present invention utilizes a tissue-altering molecule for deliveryto an ocular tissue, in specific embodiments for treatment in the eye.The tissue-altering molecule may be diffusion-enhancing, degradative, orboth, and it preferably alters the physical, chemical, or bothproperties of the tissue. A diffusion-enhancing molecule acts toincrease diffusion across Bruch's membrane by either a) reducing thethickness of the membrane itself; and/or b) reducing the amount ofdeposits within Bruch's membrane, and/or by changing the chemical natureof Bruch's membrane. It is preferably inactive upon administration tothe individual and active upon exposure to an energy source. In specificembodiments, the tissue-altering molecule is caged. In other specificembodiments, the tissue-altering molecule is a degradative enzyme, suchas cholesterol esterases, lipases, matrix metalloproteinases, or anyenzyme, or protein in particular, such as that can increase diffusionacross Bruch's membrane, preferably by degrading one or more of itscomponents. In other specific embodiments, the tissue-altering moleculeis a detergent that can extract lipidic and non-lipidic deposits fromwithin Bruch's membrane, which will increase diffusion across Bruch'smembrane.

Some tissue-altering molecules comprise at least one amino acid residueand are in inactive form by caging, wherein at least one amino acidsidechain, such as from cysteine, aspartate, glutamate, histidine,lysine, asparagine, glutamine, arginine, serine, threonine, tyrosine, ora combination thereof, comprises a photo-removable protecting group,such as at least one coumarinyl, quinoline-2-onyl, xanthenyl,thioxanthenyl, elenoxanthenyl and anthracenyl, and/or stilbenyl group.In another embodiment, the tissue-altering molecule is inactivatedthrough caging in an ultrasound contrast agent, such as microbubbles orliposomes. There are other tissue-alternating molecules without aminoacid residues known in the art, such as surfactants.

The tissue-altering molecule is formulated so as to provide an effectiveconcentration in the desired tissue. Although in some embodiments thetissue-altering molecule accumulates in non-affected tissue, this is notproblematic for the individual, since precise targeting of theactivating energy source to Bruch's membrane renders selectiveactivation within the membrane. Other regions where the cagedtissue-altering molecules accumulate are not treated with the activatingenergy; therefore, the caged tissue-altering molecules remain inactiveand are eliminated via the kidneys and/or liver. In a specificembodiment, the caged molecule is not harmful or toxic in any manner andis nevertheless excreted from the body, preferably less than about 48hours after administration, and more preferably less than about 24 hoursafter administration.

In some embodiments, the tissue-altering molecule is coupled to aspecific binding ligand that may bind to a specific target moleculewithin Bruch's membrane. The target molecule may be endogenous toBruch's membrane, or may be selectively delivered to Bruch's membrane bycrosslinking the target molecule using 2-photon irradiation. In theseembodiments, the tissue-altering molecule will be delivered in higherconcentrations to the target tissue. In a specific embodiment, variousprotein-binding domains such as leucine zipper domains are associatedwith the tissue-altering molecule.

IV. Formulations

The tissue-altering molecule is formulated so as to provide an effectiveconcentration in the desired tissue. Although in some embodiments thetissue-altering molecule accumulates in non-affected tissue, this is notproblematic for the individual, since precise targeting of theactivation energy source to Bruch's membrane renders selectiveactivation within this tissue. Other regions where the cagedtissue-altering molecules accumulate are not treated with the activatingenergy; therefore, the caged tissue-altering molecules remain inactiveand are eliminated via the kidneys and/or liver. In some embodiments,the tissue-altering molecule is coupled to a specific binding ligandthat may bind to a specific surface component of the target Bruch'smembrane or, if desired, by formulation with a carrier that delivershigher concentrations to the target tissue. In a specific embodiment,various protein binding domains such as leucine zipper domains areassociated with the tissue-altering molecule.

The nature of the formulation will depend in part on the mode ofadministration and on the nature of the selected degradation molecule.Any pharmaceutically acceptable excipient, or combination thereof,appropriate to the particular tissue-altering compound may be used.Thus, the compound may be administered as an aqueous composition, as atopical composition, as a transmucosal or transdermal composition, in anoral formulation or intravenous formulation, in a local injection (suchas periocular or intraocular) or a combination thereof. The formulationmay also include liposomes.

V. Administration and Dosage

The tissue-altering molecule compound can be administered in any of awide variety of ways, for example, orally, parenterally, or rectally, orthe compound may be placed directly in the eye, such as topically or byperiocular injection. Parenteral administration, such as intravenous,intramuscular, or subcutaneous, is useful. Intravenous, periocular, andintraocular injection are particular embodiments for delivery of thepresent invention or components thereof.

The dose of tissue-altering molecule can vary widely depending on themode of administration; the formulation in which it is carried, such asin the form of liposomes; or whether it is coupled to a target-specificligand, such as an antibody or an immunologically active fragment. As isgenerally recognized, there is a nexus between the type oftissue-altering molecule, the formulation, the mode of administration,and the dosage level. Adjustment of these parameters to fit a particularcombination is possible and routine.

VI. Energy Source

The energy source comprises any stimulus that renders an inactivetissue-altering molecule active. This preferably leads to activation ofthe inactive tissue-altering molecule. Although energy sources are wellknown in the art, exemplary forms of energy sources include light orultrasound. In specific embodiments, 2-photon photochemistry isutilized. In a specific embodiment, monochromatic light is utilized.

The various parameters used for effective, selective photo-activation ofthe tissue-altering molecules in the invention are interrelated.Therefore, the dose should also be adjusted with respect to otherparameters, for example, fluence, irradiance, duration of treatment, andtime interval between administration of the dose and the therapeuticirradiation. All of these parameters should be adjusted to produceenhancement of visual function without significant damage to the oculartissue, and a skilled artisan is well aware how to do so.

Compositions and methods related to two-photon absorption are well knownin the art, although exemplary methods are described in U.S. Pat. No.6,267,913, U.S. Pat. No. 6,472,541, and WO 00/31588, which are allincorporated by reference herein in their entirety.

VII. Generating Proteins Having Sidechains with Photo-removableProtecting Groups

Proteins may be caged using a number of strategies. Caging may beaccomplished by treating the native, uncaged molecule with a reactiveprecursor to a caging group. For example, the sidechain of the aminoacid cysteine may be caged with the photo-removable o-nitrobenzyl groupby treating a cysteine-containing protein with o-nitrobenzylbromide.Alternative strategies for caging proteins include chemical synthesis ofthe protein using solid-phase peptide synthesis starting with theappropriate caged amino acids, by direct translational incorporationinto proteins using methods based on nonsense suppression, or bysupplementing auxotrophic strains of bacteria with the caged aminoacids.

In a specific embodiment, a tissue-altering molecule is caged to renderit inactive, prior to localization to Bruch's membrane and activationupon exposure to an energy source. In a specific embodiment, thetissue-altering molecule is a protein having amino acid side chains.Those amenable to modification with a protecting group, such as aphoto-removable protecting groups, include cysteine, aspartate,glutamate, histidine, lysine, asparagine, glutamine, arginine, serine,threonine, or tyrosine. Examples of photo-removable protecting groupsincludes o-nitrobenzyl, desyl, phenacyl, trans-o-cinnamoyl, coumarinyl,quinoline-2-onyl, xanthenyl, thioxanthenyl, selenoxanthenyl andanthracenyl, stilbenyl, and/or derivatives thereof. These protectinggroups are added to the side chains as described elsewhere herein.

VIII. Macular Degeneration

Light enters through the clear front surface of the eye (the cornea),passes through the opening of the pupil, through the lens and finally isperceived by the retina in the back of the eye. The retina is amultilayered structure that lines the inside of the globe. It is made upof specialized cells that convert the light to electrical impulses thattravel to the brain and produce sight.

In the center of the retina, there is a tiny, extremely specialized areacalled the macula. It is approximately ⅛ inch in diameter-about the sizeof this letter “O”. The macula has the most densely packedphotoreceptors, which are cells that collect light. They consist of rodsand cones, which also perceive color. The macula is supplied withoxygen-rich blood that nourishes the cells.

If the macula is intact, an individual sees the fine details of whateveris directly in front of him. Macular degeneration involves thedeterioration or breakdown of this tiny structure. Central visionbecomes blurred or disappears, and straight lines look wavy or broken.The edges of images are seen, but not what is in the middle of theimage. In time, the sense of color is diminished because the cones aredamaged. However, the patient does not experience total blindness, andthere is almost always a ring of peripheral vision.

IX. Drusen

The primary characteristic of atrophic AMD is accumulation of maculardrusen, a localized thickening of Bruch's membrane. Diffuse thickeningof Bruch's membrane (basal linear deposit) is the best histopathologicpredictor of choroidal neovascularization. The drusen are primarilycomprised of vesicular material (lipids) and cross-linked protein

The presence of drusen is a common characteristic of maculardegeneration. In a specific embodiment, an individual having at leastone eye with drusen or a thickened Bruch's membrane is treated withmethods described herein.

EXAMPLES

The following examples are offered by way of example, and are notintended to limit the scope of the invention in any manner.

Example 1 Caged Tissue-Altering Molecules

Caged tissue-altering enzymes are constructed by masking various aminoacid sidechains within the protein using photo-removable protectinggroups. Examples of such groups are the o-nitrobenzyl, desyl, phenacyl,trans-o-cinnamoyl, coumarinyl, quinoline-2-onyl, xanthenyl,thioxanthenyl, selenoxanthenyl and anthracenyl, stilbenyl andderivatives thereof. These groups are introduced into the proteins bytotal chemical synthesis (including native chemical ligation), nonsensesuppression methods, or post-translational modification. Based on theknown matrix components of Bruch's membrane, cholesterol esterases,lipases, matrix metalloproteinases degrade portions of Bruch's membraneand improve trans-membrane diffusion. “Caging” these enzymes inactivatesthem. Thus, following systemic administration they circulate, bind totissues and then are eliminated in their inactive form. Application ofprecisely focused light energy to Bruch's membrane (2-photonphotochemistry) enables removal of the “cage protecting group” andselective activation of the enzyme(s) within Bruch's membrane. Onceactivated, controlled degradation takes, place and trans-membranediffusion is enhanced. This reduces the likelihood of developingchoroidal neovascularization. It also may improve dark adaptation andnight vision in elderly individuals with thickened Bruch's membrane. Italso may cause regression of established choroidal neovascularization.

Alternatively, mild detergent molecules (e.g. surfactant) are “caged”using groups such as the o-nitrobenzyl, desyl, phenacyl,trans-o-cinnamoyl, coumarinyl, quinoline-2-onyl, xanthenyl,thioxanthenyl, selenoxanthen and anthracenyl, and/or stilbenyl moietiesand their derivatives thereof and likewise activated by irradiation (forexample, by 2-photon irradiation) to effect selective biochemicalmodification of Bruch's membrane. Similar to enzyme treatment, theseactivated detergent molecules alter the diffusion barrier of agedBruch's membrane to lessen the likelihood of visual loss and/or improvevisual function.

Example 2 Alternative Inactivation Embodiments

Degradative enzymes (matrix metalloproteinases, cholesterol esterases,lipases, serine proteases) can also be incorporated into ultrasoundcontrast agents such as microbubbles and liposomes. Carried within thesecontrast agents, the degradative molecules are inactive. Preciseapplication of ultrasonic energy to Bruch's membrane causes cavitationsof the contrast agent and release of the degradative enzymes intoBruch's membrane. Similar to the example above, enzymatic releaseimproves trans-membrane diffusion and potentially improves visualfunction. Not activated by ultrasound, remaining extraocularencapsulated inactive enzymes are eliminated from the body withoutcausing collateral tissue alterations.

Example 3 Targeting Improvements

Many of the enzymes administered in the previous examples are allpresent in Bruch's membrane and/or the RPE. Further, they are present inthe circulation. To increase the local concentration of these enzymeswithin Bruch's membrane, these enzymes may be fused to various proteinbinding domains, such as a leucine zipper domain, a chitin-bindingdomain, or a Src homology 2 (SH2) domain. A skilled artisan recognizesthat hetero-dimeric zippers consist of an acidic and a basic partner andmay self assemble into coiled-coils that exist as dimers and higherorder aggregates. Thus, in some embodiments, an acidic (basic) leucinezipper domain may be selectively delivered to Bruch's membrane viaphoto-crosslinking initiated by 2-photon irradiation. In specificembodiments, the enzyme is fused to a basic (acidic) leucine zipperdomain and is administered, for example, systemically (such as orally orintravenously), or by injection (such as intraocular and/or periocularinjection) and distributed to extracellular tissues, including Bruch'smembrane. Formation of hetero-dimers within Bruch's membrane increasesthe local concentration of degradative enzymes and subsequently enhancestrans-membrane diffusion properties. Because the concentration of thesedegradative enzymes will remain low in other tissues, there are noundesired collateral tissue alterations. Additionally, enzymes may beproduced containing photo-active groups (such as the benzophenoyl,phenylazide, trifluromethylphenyldiazirinyl, and derivatives thereof)using the methods outlined in Example 1. Systemic administration of suchenzymes, followed by selective irradiation of Bruch's membrane initiatesphoto-crosslinking between the enzymes and Bruch's membrane, increasingthe concentrations of the degradative enzymes. In other embodiments, theprotein-binding domains facilitate association of a targetingtissue-altering molecule with the tissue to be targeted, such as bybinding to proteins on or within the tissue.

Example 4 Treatment of Ocular Disorder

A patient with an ocular disorder, such as atrophic age-related maculardegeneration (in exemplary embodiments), is administered a caged,inactivated enzyme capable of altering Bruch's membrane uponphotoactivation. In specific embodiments, the administration is systemic(such as intravenously or orally), or by injection (such as intraocularand/or periocular injection). In other specific embodiments, the caged,inactivated enzyme is labeled, such as fluorescently labeled, althoughin alternative embodiments the caged, inactivated enzyme is not labeled.

For illustrative purposes only, the caged, inactivated molecule isfluorescent. Several minutes following administration, the fluorescent,caged degradative complex is viewed in macular Bruch's membrane by 2photon irradiation. After focusing on the fluorescent label in macularBruch's membrane, a higher dose of 2 photon irradiation is applied touncage the enzyme and initiate partial degradation of Bruch's membrane.Systemically distributed non-irradiated caged enzyme is excreted in itsinactive form. Two photon irradiation viewing of labeled Bruch'smembrane and photoactivation of the degradative substance are bothperformed at levels non-toxic to ocular structures.

Alternatively, a fluorescent label not attached to the caged degradativeenzyme complex is administered systemically. About ten to sixty minuteslater, the caged degradative molecular complex is administered. Aboutfive minutes later, Bruch's membrane is visualized by 2 photonirradiation of the fluorescent label. A higher dose of 2 photonirradiation is then used to activate the caged degradative molecularcomplex.

In another embodiment, a fluorescent label not attached to the cageddegradative enzyme complex is administered systemically. About ten tosixty minutes later, 2 photon irradiation is used to visualize thequantity of fluorescence in Bruch's membrane. Quantification offluorescence is a diagnostic indicator of severity of atrophic maculardegeneration.

In another embodiment of the present invention, there is a method ofidentifying a tissue to be treated based on the intrinsic lightscattering from targeted tissue such as Bruch's membrane. Opticalcoherence tomography (OCT) with visible or infrared light is used todetect alterations in the physical or chemical nature of Bruch'smembrane in the eye. OCT can be used to see not only the structure inthe eye but also the mobility of the structures by Doppler OCT and thechemical nature by combining the OCT with an exogenous dye. In thisembodiment, the OCT and/or its variants are used to determine the natureof the Bruch's membrane with altered properties to permit guidedtreatment. Treatment could be the photo-uncaging or photo-activation orphoto-ablation of intrinsic or extrinsic substances in or near Bruch'smembrane.

An exemplary embodiment targeting Bruch's membrane is depicted in FIG.1.

REFERENCES

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referenceherein.

-   U.S. Pat. No. 5,756,541-   U.S. Pat. No. 5,798,349-   U.S. Pat. No. 5,910,510-   U.S. Pat. No. 5,935,942-   U.S. Pat. No. 6,128,525-   U.S. Pat. No. 6,140,314-   U.S. Pat. No. 6,225,303-   U.S. Pat. No. 6,248,727-   U.S. Pat. No. 6,267,913-   U.S. Pat. No. 6,472,541-   WO 00/31588

1. A method of treating dry macular degeneration in the eye of anindividual, comprising administering a caged detergent to theindividual; and selectively applying two-photon irradiation to the cageddetergent in Bruch's membrane to activate the detergent, resulting in anincrease in diffusion across the membrane.
 2. The method of claim 1,wherein the detergent is further defined as being caged by a compoundcomprising at least one o-nitrobenzyl, desyl, phenacyl,trans-o-cinnamoyl, coumarinyl, quinoline-2-onyl, xanthenyl,thioxanthenyl, selenoxanthenyl, anthracenyl, or stilbenyl group.
 3. Themethod of claim 1, wherein the increase in diffusion across the membraneresults from a reduction of the thickness of the membrane, an alterationof the composition of the membrane, or both.
 4. The method of claim 1,wherein the caged detergent binds directly to the membrane.
 5. Themethod of claim 1, wherein the caged detergent is administered in apharmaceutically acceptable excipient systemically to the individual. 6.The method of claim 1, wherein the caged detergent is administered in apharmaceutically acceptable excipient to the individual orally, byinjection, rectally, vaginally or topically.
 7. The method of claim 6,wherein said injection is intraocular or periocular.
 8. The method ofclaim 1, wherein the caged detergent is labeled with a fluorescentmolecules.
 9. The method of claim 1, wherein said method furthercomprises the step of visualizing said membrane.
 10. The method of claim9, wherein said membrane visualizing is achieved by delivery of atargeted fluorescent label to the membrane, by identification of themembrane's inherent autofluorescence, or by optical coherence tomography(OCT) Doppler.
 11. The method of claim. 1, wherein the increase indiffusion across the membrane is due to an alteration of a chemicalproperty of Bruch's membrane, of a physical property of Bruch'smembrane, or both.
 12. The method of claim 1, further comprising thestep of visualizing Bruch's membrane by optical coherence tomography(OCT) Doppler.